The glutamate receptor gene family encodes ligand-gated ion channels that can be divided into three classes ((AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), kainate, and NMDA (N-methyl-D-aspartic acid)) on the basis of agonist pharmacology and molecular structure (Dingledine et al. 1999; Qian & Johnson 2002; Erreger et al 2004; Wollmuth & Sobolevsky 2004). NMDA receptors mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system, and have garnered considerable attention because of their prominent role in many normal brain functions, including synaptic plasticity (Lisman 2003; Miyamoto 2006), frequency encoding of information (Froemke et al 2005; Kampa et al 2006; Rhodes 2006), and neuronal development (Rudhard et al 2003; Colonnese et al 2005, 2006; Waters & Machaalani 2005; Nacher & McEwen 2006). In addition, NMDA receptors play an overt role in neuropathology of ischemia and traumatic brain injury (Whetsell 1996; Miyabe et al 1997; Dirnagl et al 1999; Brauner-Osborne et al 2000; Wang & Shuaib 2005). NMDA receptors have been suggested to be involved in a wide range of neurological diseases, including schizophrenia, depression, psychosis, Huntington's disease, Alzheimer's disease, and Parkinson's disease.
NMDA receptors are tetrameric complexes comprised of glycine-binding NR1 subunits, glutamate-binding NR2 subunits, and NR3 (A and B) subunits. The subunit composition determines the functional properties of native NMDA receptors.
Expression of the NR1 subunit alone does not produce a functional receptor. Co-expression of one or more NR2 subunits or one or more NR3 subunits is required to form functional channels. In addition to glutamate, the NMDA receptor requires the binding of a co-agonist, glycine, to allow the receptor to function. A glycine binding site is found on the NR1 and NR3 subunits, whereas the glutamate binding site is found on NR2 subunits. The four NR2 subunits (NR2A, B, C, and D) each endow the receptor with surprisingly divergent single channel conductances, deactivation time courses, and open probabilities (Stern et al 1992; Wyllie et al 1998; Vicini et al 1998; Erreger et al 2004; Erreger et al 2005ab). The increasingly precise anatomical localization of the NR2 subunits (Akazawa et al 1994; Monyer et al 1994; Buller et al 1994; Paquet et al 1997; Dunah et al 1998; Thompson et al 2002; Lau et al 2003; Lopez de Armentia & Sah 2003; Dunah & Standaert 2003; Dunah et al 2003; Hallett & Standaert 2004; Salter & Fern 2005; Karodottir et al 2005) has strengthened the therapeutic rationale for the development of subunit-selective NMDA receptor potentiators, which should target NMDA receptor functions in specific brain regions without engaging NMDA receptors elsewhere. This idea has fueled optimism that NR2 subunit-selective modulators might be well-tolerated therapeutic agents for a wide variety of different indications.
At resting membrane potentials, NMDA receptors are largely inactive due to a voltage-dependent block of the channel pore by magnesium ions. Depolarization releases this channel block and permits passage of calcium as well as other cations. NR2A- and NR2B-containing NMDA receptors are more sensitive to Mg2+ blockade than NR2C- and NR2D-containing receptors. The NMDA receptor is modulated by a number of endogenous and exogenous compounds, including, sodium, potassium, and calcium ions that can not only pass through the NMDA receptor channel but also modulate the activity of receptors. Zinc blocks the channel through NR2A- and NR2B-containing receptors in a noncompetitive and voltage-independent manner. Polyamines can also either potentiate or inhibit glutamate-mediated responses (See, for example, McGurk et al., Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 9971-9974, December 1990).
NR2-Subunit Selectivity of Existing NMDA Receptor Modulators
Few NMDA receptor potentiators (or positive modulators) have been described to date in the literature. Perhaps the best known potentiators are naturally occurring polyamines and neurosteroids. Extracellular polyamines such as spermine and spermidine can potentiate with low potency (EC50 100's μM) the function only of NR2B-containing NMDA receptors (Williams et al 1994; Traynelis et al 1995). In addition, various neurosteroids both potentiate and inhibit NR2A/B receptors, depending on concentration and subunit composition. Neurosteroids bind at much higher rates to closed receptors than active receptors (Horak et al 2004, 2006). It is believed that highly subunit-selective drug-like potentiators of heterodimeric NMDA receptors containing NR2A, NR2B, NR2C, or NR2D subunits are heretofore unknown. Experience with non-selective and subunit selective NMDA receptor antagonists suggests that subunit selective potentiators will likely have fewer side effects than non-selective potentiators that act at all NMDA receptors. Moreover, because subunits show differential distribution in the brain, it stands to reason that identifying compounds that target specific subunits may bring about a therapeutically useful effect in one brain region while minimizing effects in brain regions that lack that particular subunit.
Clinical Relevance of NMDA Receptor Potentiators: Learning and Memory
Enhancement of NMDA receptor activity has been proposed to be a useful therapeutic strategy for certain conditions associated with altered cognitive function (Lisman et al., 2008). Overexpression of the NR2B subunit can enhance learning and memory in animal models (Tang et al., 1999, 2001; Cao et al., 2007). In addition, D-cycloserine has been studied as an adjunct to behavioral therapy to promote the extinction of maladaptive associations. This approach is based on the hypothesis that D-cycloserine will augment or enhance therapy-directed learning through potentiation of NMDA receptor-dependent learning. This potentiation is believed to occur through the increased D-serine occupancy of NR1 glycine binding sites. D-cycloserine increased the efficacy of behavioral therapy in clinical trials involving patients suffering acrophobia (Ressler et al., 2004), social anxiety disorder (Hofmann et al., 2006), or obsessive compulsive disorder (Kushner et al., 2007; Wilhelm et al., 2008). The complex multi-subunit composition of NMDA receptors offers further opportunities for pharmacological manipulation for therapeutic gain while minimizing side effects by allowing therapy to be targeted at brain regions expressing specific NR2 subunits.
Clinical Relevance of NMDA Receptor Potentiators for Schizophrenia, Psychoses, Bipolar Disorder, and Depression
Schizophrenia and psychosis and other neuropsychiatric disorders arise from changes in neurotransmitter systems, neuronal connectivity, or both. The dopamine hyperactivity hypothesis for schizophrenia, supported by years of clinical experience and neurochemical data, maintains that overactivation of dopamine receptors, such as the D2 subtype, leads to cognitive dysfunction that can be treated by competitive dopaminergic antagonists (Hirsch & Barnes 1995; Seeman et al 2006). The recognition that NMDA inhibitor-induced behavioral effects closely mimic the symptoms of schizophrenia (Javitt and Zukin, 1991; Luby et al., 1959) lead to the hypothesis that NMDA receptor hypofunction may be a causative factor in schizophrenia (Javitt, 2007; Krystal et al., 2002; Olney et al., 1999; Tsai and Coyle, 2002; Yamada et al., 2005; Morita et al., 2007). That is, if blockade of NMDA receptors can reproduce schizophrenic symptoms, it stands to reason that hypofunction of the NMDA receptor system might underlie these symptoms (Coyle et al 2003). This hypothesis led to the proposal that potentiation of NMDA receptor function may have therapeutic benefit in patients suffering from schizophrenia (Heresco-Levy, 2000; Morris et al 2005).
Circuit-based models of schizophrenia have further highlighted the potential role of NMDA receptor hypofunction in interneurons (Lisman et al 2008), which happen to express NR2C and/or NR2D subunits (Monyer et al 1994; Rudolph et al 1996; Thompson et al 2002; Binshtok et al 2006). These models predict that enhancement of interneuron activity (for example by NR2C/D selective potentiators) could be beneficial for patients. Similarly, the ability of certain NR2B-selective antagonists to produce psychosis (Preskorn et al 2008) suggests that potentiation of NMDA receptors containing the NR2B subunit may also be therapeutically beneficial. While not wishing to be bound to a particular theory, such subunit-selective potentiators might bind to a site independent of the agonist recognition site, and enhance the proportion of time the receptor remains open when agonists such as glutamate and glycine bind. Thus, the regional and cell-specific differences in the expression of the NR2C and NR2D subunits in interneurons provides a rationale for the development of NR2C/D-selective potentiators that may improve negative and cognitive symptoms, or influence mood. Additional support for this idea is derived from the observation that enhancement of NMDA receptor function with glycine-site agonists and glycine transport inhibitors may improve negative and cognitive symptoms when used as adjuncts to current antipsychotic therapies (Javitt et al 1994; Depoortere et al 2005). This finding provided rationale for clinical trials of agonists at the glycine site on the NMDA receptor (Coyle and Tsai, 2004; Labrie and Roder, 2009; Shim et al., 2008). Several studies of the use of glycine and D-serine as adjuncts to antipsychotics therapy revealed moderate reduction of negative symptoms and suggested a trend toward a decrease in cognitive symptoms (Tuominen et al., 2005). One subsequent clinical trial suggested a beneficial effect of the glycine site agonist D-alanine on both positive and negative symptoms of schizophrenia (Tsai et al., 2006). D-cycloserine, an antibiotic and glycine site ligand, is a partial agonist at the glycine site and preferentially activates NMDA receptors containing the NR2C subunit (Sheinen et al., 2001; Dravid et al., 2010). Initial clinical studies of D-cycloserine indicated a beneficial effect on negative symptoms (Goff et al., 1999). Because preclinical data suggest the possibility of tachyphylaxis to glycine site ligands, D-cycloserine has been examined with intermittent dosing, which improved negative symptoms in patients suffering schizophrenia (Goff et al., 2008a). Thus, there is general optimism that if allosteric potentiators of NMDA receptor function could be found, such compounds might provide beneficial effects by reducing NMDA receptor hypofunction in psychoses and schizophrenia (Heresco-Levy 2005; Lindsley et al 2006), or perhaps by having antidepressant effects.
Additional circumstantial and correlative data are consistent with a role for NR2C/D subunit in schizophrenia, psychoses, and neuropsychiatric conditions. NR2D subunit mRNA is significantly increased in the prefrontal cortex of schizophrenic patients (Akbarian et al 1996), and NR2D protein expression increases in the frontal cortex of PCP-treated rats (Lindahl & Keifer 2004). Because NMDA receptor hypofunction in frontal and prefrontal cortex correlates with negative symptoms and cognitive impairments (Andreasen et al 1997; Molina et al 2005), an NR2D potentiator might be a useful therapy to treat these symptoms. Interestingly, genetic analysis of polymorphisms suggests that the NR2D gene may be a locus contributing to schizophrenia susceptibility in the Japanese population (Makino et al 2005).
Clinical Relevance of NMDA Receptor Potentiators: Facilitation of Motor Learning During Rehabilitation
Of the 1.4 million people who sustain a traumatic brain injury (TBI) each year in the United States, 50,000 die, 235,000 are hospitalized, and 1.1 million are treated and released from an emergency department (Langlois et al., 2006). The Centers for Disease Control and Prevention estimates that at least 5.3 million Americans currently have a long-term or lifelong need for help to perform activities of daily living as a result of a TBI (Thurman et al 1999). On average, every 45 seconds someone in the United States has a stroke, giving rise to 700,000 cases of stroke every year, of which 75% are likely to survive with impaired function requiring rehabilitation (Jorgensen et al., 1995). Millions of stroke and TBI survivors thus suffer from a movement-related problem. Together, stroke and TBI have a greater disability impact than virtually all other neurological conditions and chronic diseases.
The exceptionally large number of patients suffering from disabilities creates a strong need to develop new treatments to facilitate recovery of cortical function following acute neural insults, as occur in ischemic conditions, stroke and traumatic brain injury. Accomplishment of this task has the potential to improve clinical outcomes and quality of life for patients suffering brain injury, stroke, hypoxia, or ischemia. Several recent studies involving NMDA receptors suggest a path towards development of new therapies to enhance cortical motor learning and facilitate recovery from brain insult (See, for example, Nitsche et al., Neuropsychopharmacology (2004) 29, 1573-1578). NMDA receptors require the simultaneous binding of two ligands (glycine, glutamate) before they open to initiate depolarizing current flow into a neuron. A clinically approved partial agonist at the glycine site of the NMDA receptor (D-cycloserine) influences emotional learning, being a potentiator of extinction of conditioned fear in both animal models and human anxiety disorders (Walker et al., 2002; Ressler et al., 2004; Hofmann et al., 2006ab). If motor learning is amenable to pharmacological manipulation in the same manner as emotional learning, this may provide a means to improve rehabilitation of patients suffering neuronal loss as a consequence of stroke or TBI through enhancement of NMDA receptor function during physical therapy.
While the molecular basis for the behavioral effects of D-cycloserine has not been elucidated, several clues exist as to why D-cycloserine might have unique behavioral actions that other partial or full agonists at the either the glycine or glutamate binding site on the NMDA receptor appear to lack. NMDA receptors are comprised of NR1 and NR2 subunits, and D-cycloserine at maximally effective concentrations appears to cause slightly lower responses than maximally effective levels of glycine (the endogenous ligand) at NMDA receptors comprised of NR1/NR2A, NR1/NR2B, and NR1/NR2D subunits. By contrast, a recent study demonstrated that D-cycloserine causes current responses at NR1/NR2C receptors that are nearly twice as large as the endogenous agonist glycine (Sheinin et al., 2002). That is, the agonist D-cycloserine appears to selectively enhance NMDA receptor function when the NR2C subunit is present through its binding to the glycine recognition site on the NR1 subunit. This finding suggests that the unique behavioral effects of D-cycloserine may be related to the potentiation of NR2C-containing NMDA receptors. Implicit in this hypothesis is the idea that enhancement of only NMDA receptors that contain the NR2C subunit may enhance emotional learning.
In cortical structures (hippocampus and neocortex), NR2C subunit mRNA is expressed in subsets of interneurons (Monyer et al., 1994; Binshtock et al., 2006), suggesting that modulation of NR2C function has the potential to sculpt network activity through modulation of interneuronal firing. Thus, NR2C potentiators may be useful as cognitive enhancers, with many potential functions, including treatment and prevention of neurodegenerative diseases associated with cognitive decline. In addition, subunit selective NMDA receptor potentiation may be useful for improving rehabilitation, for example, from stroke and traumatic brain injury.
Clinical Relevance of NMDA Receptor Potentiators: Modulation of Motor Function
NMDA receptors containing the NR2C subunit are highly expressed in cerebellum (Monyer et al 1994; Lansola et al., 2005), a structure well known to be important for sculpting motor function, in particular coordination and fine motor movement. NR2C is particularly abundant at the mossy-fiber-granule cell synapse, and thus modulators of NR2C may have effects on cerebellar function through actions at this synapse, which ultimately gives rise to the input to Purkinje cells via the parallel fibers. In addition, NR2D subunits have been proposed to be expressed by neurons of the deep cerebellar nuclei (Cull-Candy et al., 1998), providing another target for influencing cerebellar function. Thus, NR2C/D-selective NMDA receptor potentiators may control information processing within the cerebellum, and thus have useful effects on motor function, coordination, motor learning, or movement control. Therefore, NR2C/D potentiators can be used to treat a wide range of neurological diseases associated with impaired motor function.
Clinical Relevance of NMDA Receptor Potentiators: Epilepsy
Interneurons typically utilize the inhibitory neurotransmitter GABA and contact a large number of cells. Interneuron firing thus has the ability to hyperpolarize large numbers of neurons. In this way, interneurons can have far-reaching effects on neuronal excitability and signal processing in the central nervous system. Epilepsy is a disorder associated with hypersynchronous and excessive neuronal firing, giving rise to both electrographic and motor seizures. Interneuron inhibition is thought to limit excessive tissue excitability, and a number of compounds that enhance GABA receptor function (e.g. phenobarbital, benzodiazepine) are useful as anticonvulsant agents in some settings. Because NR2C- and NR2D-containing receptors are expressed in hippocampal and cortical inhibitory interneurons but not excitatory principle cells (Monyer et al 1994; Rudolph et al 1996; Thompson et al 2002; Binshtok et al 2006), modulators that selectively enhance NR2C and NR2D receptor function should depolarize interneurons, and thereby increase firing of GABAergic interneurons. As interneurons fire more action potentials, the resulting release of GABA onto excitatory principle cells exerts an inhibitory effect that can be anticonvulsant. Thus, NR2C and NR2D potentiators can be used for their anticonvulsant properties.
Treatment of Bone Disorders
NMDA receptors of the NR2D subtype are found in the osteoblasts, and therefore, compounds which have activity at these receptors can be useful in treating bone disorders.
The bone-remodeling cycle occurs at particular areas on the surfaces of bones. Osteoclasts which are formed from appropriate precursor cells within bones resorb portions of bone; new bone is then generated by osteoblastic activity. Osteoblasts synthesise the collagenous precursors of bone matrix and also regulate its mineralization. The dynamic activity of osteoblasts in the bone remodelling cycle to meet the requirements of skeletal growth and matrix and also regulate its maintenance and mechanical function is thought to be influenced by various factors, such as hormones, growth factors, physical activity and other stimuli. Osteoblasts are thought to have receptors for parathyroid hormone and estrogen. Ostoeclasts adhere to the surface of bone undergoing resorption and are thought to be activated by some form of signal from osteoblasts.
Irregularities in one or more stages of the bone-remodelling cycle (e.g. where the balance between bone formation and resorption is lost) can lead to bone remodelling disorders, or metabolic bone diseases. Examples of such diseases are osteoporosis, Paget's disease and rickets. Some of these diseases are caused by over-activity of one half of the bone-remodelling cycle compared with the other, i.e. by osteoclasts or osteoblasts. In osteoporosis, for example, there is a relative increase in osteoclastic activity which may cause a reduction in bone density and mass. Osteoporosis is the most common of the metabolic bone diseases and may be either a primary disease or may be secondary to another disease or other diseases.
Post-menopausal osteoporosis is currently the most common form of osteoporosis. Senile osteoporosis afflicts elderly patients of either sex and younger individuals occasionally suffer from osteoporosis.
Osteoporosis is characterized generally by a loss of bone density. Thinning and weakening of the bones leads to increased fracturing from minimal trauma. The most prevalent fracturing in post-menopausal osteoporotics is of the wrist and spine. Senile osteoporosis, is characterized by a higher than average fracturing of the femur.
The tight coupling between the osteoblastic and osteoclastic activities of the bone remodeling cycle make the replacement of bone already lost an extremely difficult challenge. Consequently, research into treatments for prevention or prophylaxis of osteoporosis (as opposed to replacement of already-lost bone) has yielded greater results to date.
Estrogen deficiency has been considered to be a major cause of post-menopausal osteoporosis. Indeed steroids including estrogen have been used as therapeutic agents (New Eng. J. Med., 303, 1195 (1980)). However, recent studies have concluded that other causes must exist (J. Clin. Invest., 77, 1487 (1986)).
Other bone diseases can be caused by an irregularity in the bone-remodeling cycle whereby both increased bone resorption and increased bone formation occur. Paget's disease is one such example.
There remains a need for improved neuroprotective compounds and methods for the treatment of neuropathologies that have reduced toxicity. There is also a need for improved treatments for neuropathic pain, inflammatory pain, stroke, traumatic brain injury, global ischemia, hypoxia, spinal cord trauma, epilepsy, addiction, depression, schizophrenia, motor disorders, and neurodegenerative diseases and disorders.
It would be advantageous to have compounds, compositions including the compounds, and methods of treatment using the compounds to treat these disorders. The present invention provides such compounds, compositions, and methods of treatment.